The long term goals of this application are to define molecular events that regulate NK cell development and function, thereby providing insight into pathways that may be manipulated to promote or inhibit these aspects of NK cell biology. NK cells are innate immune lymphocytes that are important during host defense against infections and mediate anti-neoplastic immune responses. This is exemplified by rare patients with selective NK cell deficiencies that succumb to fatal herpesvirus infections early in life, demonstrating the critical importance of these cells for human health. Moreover, allogeneic NK cells can be an effective treatment for acute myeloid leukemia patients, and thus NK cells hold promise as a cancer immunotherapy approach. Currently, we have an incomplete understanding of the molecular mechanisms responsible for regulating NK cell functionality (cytokine production, cytotoxicity, proliferation). MicroRNAs (miRNAs) are small regulatory RNAs that target mRNA stability and/or limit protein translation, consequently regulating critical cellular processes. Recent studies have identified miRNAs expressed in resting and activated mouse and human NK cells, and have shown that global miRNA deficiency results in altered mature NK cell functional responses in vitro and in vivo. However, our understanding of how individual miRNAs regulate NK cell biology is limited. Of multiple candidate miRNAs that are expressed and have a rationale for regulating NK cell function, miR-155 was prioritized at the highest for in depth study. This application describes a 5 year plan to study the regulatory role o miR-155 on NK cell function. We hypothesize that miR-155 regulates key aspects of NK cell functionality, including effector cytokine (e.g., IFN-?) production, cytotoxicity, and intracellula pathways important for promoting or inhibiting NK cell activation. Our approach utilizes NK cell-specific (Ncr1-iCre) conditional gain (via loxP-STOP-loxP overexpression) and loss (floxed) of function models for miR-155. In Aim 1 miR-155 mouse models will be used to define the regulatory contribution of miR-155 to NK cell function, including development/maturation, IFN-? production, and cytotoxicity. In Aim 2 we will elucidate the regulatory mechanism(s) of miR-155 by defining their target mRNAs in NK cells using bioinformatics, mRNA profiling, and Argonaute-immunoprecipitation followed by next-generation sequencing. These targets will in turn be assessed in functional experiments, described in Aims 1 and 3, and confirmed in human NK cells. In Aim 3 we will define the in vivo significance of miR-155 regulation on NK cell function using model pathogens. There is a high level of integration between Aim 2 and Aims 1 and 3, to deeply define mechanisms whereby miR-155 targets specific mRNAs, which in turn regulate NK cell function. Thus, these studies will provide novel information about how miR-155 regulates NK cell activation, their mechanism of action by identifying mRNA targets, and the importance of miR-155 to NK cell-mediated host defense in vivo. These studies may therefore inform future strategies to augment NK cell functionality as an immunotherapeutic strategy.